The present invention relates to three dimensional optical tomography using point source projection geometry, and, more particularly, to imaging microscopic objects, including biological cells, in a flow stream using optical tomography.
With the advent of molecular probes, such as antibody probes and nucleic acid hybridization probes, new disease related questions can be addressed by tagging these molecular probes and then measuring their location and concentration within biological cells and tissues. As the need to more accurately localize and quantify these probes is emerging, there is a concomitant need for improved techniques to measure probe densities microscopically in two dimensions (2D) and three dimensions (3D). Traditional light microscopy, which utilizes cells mounted on glass slides, can only approximate 2D and 3D because of limitations in focal plane depth, sampling angles, and problems with cell preparations that typically cause cells to overlap in the plane of the image. Another drawback of light microscopy is the inherent limitation of viewing through an objective lens where only the area within the focal plane provides accurate data for analysis.
Flow cytometry methods generally overcome the cell overlap problem by causing cells to flow one-by-one in a fluid stream. Unfortunately, flow cytometry systems do not generate images of cells of the same quality as traditional light microscopy, and, in any case, the images are not three dimensional. For background, those skilled in the art are directed to Shapiro, H M, Practical Flow Cytometry, 3rd ed., Wiley-Liss, 1995.
In the area of computer aided tomography, U.S. Pat. No. 5,402,460, issued Mar. 28, 1995, to Johnson, et al. entitled xe2x80x9cThree dimensional Microtomographic Analysis Systemxe2x80x9d discloses a microtomographic system for generating high-resolution, three dimensional images of a specimen using an x-ray generator and an x-ray detector that measures the attenuation of the x-ray beam through the specimen. Two projections, each using a different energy x-ray beam, of each view of the specimen are made with Johnson, et al.""s microtomographic system. After the two projections of one view of the specimen are made, the specimen is rotated on the specimen holder and another set of projections is made. The projections of each view of the specimen are analyzed together to provide a quantitative indication of the phase fraction of the material comprising the specimen. The projections of the different views are combined to provide a three dimensional image of the specimen. U.S. Pat. No. 5,402,460 is incorporated herein by reference. Although the x-ray technology as taught by U.S. Pat. No. 5,402,460 is useful for some applications, it does not provide an optical solution useful for flow cytometry.
To overcome the aforementioned limitations and others found in such systems, it is a motivation of this invention to combine the one-by-one cell presentation of flow cytometry with computational optical tomography from multiple point source projections to reconstruct 2D and 3D cell density information from a plurality of projections.
The invention provides an apparatus and method for imaging small objects in a flow stream using optical point source projections and tomographic image reconstruction. The flow optical tomography (FOT) system includes a flow cytometer, as shown in FIG. 1, and a reconstruction cylinder, as shown in FIG. 4, positioned around capillary tube 2. A source of photons 25 and a photon sensor 26 work together with pulse height analyzer 27 to operate as a triggering device. Pulse height analyzer 27 operates in accordance with known principals to provide a first trigger point 28 for the beginning of a cell, and a second trigger point 29 for the end of the cell. The pulse height analyzer 27 outputs a trigger signal 30 corresponding to the beginning and end of each cell, where the trigger signal is received by the reconstruction cylinder 20. Signals from the reconstruction cylinder may be analyzed directly or processed using computerized tomographic image reconstruction techniques to provide two or three dimensional information about cells.